Alterations in the cholinergic physiology of the brain were the first to be observed when research on environmental influences on postnatal brain development began 35 years ago. Since then, the effects of isolated rearing (IR) or early pharmacological insults have been shown not only on the physiology, but also the anatomy of a variety of transmitter systems. The cholinergic fiber density, however, still remained to be assessed. We therefore used a histochemical procedure to stain cholinergic fibers in the brains of young adult gerbils reared either in groups in enriched environments or isolated in standard makrolon cages. Half of the animals from each rearing condition had received a single high dose of methamphetamine on postnatal day 14. Fiber densities were measured by computerized image analysis in the medial and orbital prefrontal cortex (PFC), dysgranular and granular insular cortex, sensorimotor cortices, and the entorhinal cortex of both hemispheres. Isolation Tearing increased the cholinergic fiber densities in the prefrontal cortices of the left hemisphere and in the entorhinal cortex of the right hemisphere by about 10%, with no effect in the respective contralateral side. The early methamphetamine intoxication showed no influence in prefrontal and entorhinal cortices, but diminished the acetylcholine (ACh) innervation of the forelimb area of cortex in both hemispheres in IR gerbils and of the left hemisphere in ER gerbils, and reduced the acetylcholine innervation in the hindlimb area in both sides in both rearing groups. These results demonstrate that (a) cholinergic fiber density is differentially regulated in different cortical areas and (b) the plasticity of the cholinergic system can only be understood in the interplay with other neuromodulatory innervations. (C) 2004 Elsevier Inc. All rights reserved.